Precipitation methods can be classified into two groups: precipitation and coprecipitation, based on the functional mechanisms involved. Classical precipitation methods usually employ large amounts of additives that alter the characteristics of the solvent in a way that renders the species to be precipitated insoluble. The additive itself may remain soluble and is mostly removed when the supernatant is separated from the precipitate. The precipitate is then resuspended in a fluid lacking the agent that was used to mediate precipitation. Traces of the agents can be easily removed since they do not form persistent associations with the product being precipitated. Examples include precipitation with salts such as ammonium sulfate, sodium citrate, and potassium phosphate, among others; organic polymers such as polyethylene glycol, polypropylene glycol, dextran, and polyvinylpyrrolidone, among others; and organic solvents such as acetone, chloroform, and alcohols, among others.
Co-precipitation methods typically work by binding to a species to be precipitated and reducing its solubility to a point where it precipitates spontaneously. The technique can be used to selectively precipitate a protein or virus of research or commercial interest. Equally, the technique can be used to selectively precipitate one or more contaminant species from a preparation containing proteins or DNA plasmids of interest. Contaminants that are important to remove from protein and DNA plasmid preparations particularly include viruses and endotoxins. Co-precipitation is generally advantageous over classical precipitation in the sense that it usually uses lower amounts of the precipitating agent, but disadvantageous in the sense that recovery of the precipitated product employs not only its re-suspension in the absence of precipitating agent, but may impose the need for an additional processing step to displace residual precipitating agent that remains bound in trace amounts to the product of interest. This is usually done by introducing an agent that disrupts the interaction between the product and the co-precipitant. Examples of co-precipitating agents include anionic polymers, cationic polymers, and fatty acids, among others. Substances used to displace residual precipitating agent include high concentrations of neutral salts such as sodium chloride, chaotropes, and organic solvents, among others.
Both classical precipitation methods and co-precipitation methods have been used in the purification of virus. Many types of chemical surfaces have the ability to bind virus. Some include surfaces that are chemically modified to mediate interactions with viruses through positive or negative charges, or hydrophobicity, such as ion exchangers and hydrophobic interaction chromatography media. These materials have the desirable feature of binding diverse virus species and fairly low cost, but also the undesirable features of binding a great number of proteins and requiring extensive process development. Alternatively, immobilization of antibodies as bioaffinity ligands on surfaces can be specific for virus as opposed to other proteins and require only limited process development, but surfaces with immobilized antibodies typically bind only a single virus species and they are comparatively very expensive.
Ureides have exhibited activity as non-inflammatory or anti-inflammatory agents that are used widely in products for human skin care. One common example is allantoin, which is poorly soluble in aqueous solutions and saturates at a concentration of about 36 mM. Amounts above this concentration exist as crystals. Some ureides have been covalently immobilized on silica particles. Immobilization of 1-[3-(trimethoxysilyl)propyl]urea on silica has been demonstrated (Bicker et al. J. Chromatogr. A, 1218 882-895 2011), and the construct used for hydrophilic interaction chromatography of a variety of small molecule compounds. A bidentate alkoxysilane has been immobilized (Kotoni et al J. Chromatogr. A, 1232 196-211 2012) incorporating urea fragments on silica, also for hydrophilic interaction chromatography, and was found to be useful for the analysis of sugars. The use of allantoin in combination with multivalent cations for clarification of aggregates from antibody-containing cell culture supernatants has been described (J. Chromatogr. A, 1291 33-40 2013).